Recombinant polypeptide construct comprising multiple enterotoxigenic Escherichia coli fimbrial subunits
Inventors
Assignees
Publication Number
US-9328150-B2
Publication Date
2016-05-03
Expiration Date
2026-01-10
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Abstract
The inventive subject matter relates to a recombinant polypeptide construct comprising enterotoxigenic Escherichia coli fimbrial subunits. The recombinant polypeptide constructs comprise multiple subunits to the same or different ETEC fimbrial types. The constructs are useful for inclusion in immunogenic formulations for the inductin of immunity against entertoxigenic Escherichia coli. The inventive subject matter also relates to the use of the recombinant polypeptide constructs in induce anti-enterotoxigenic Escherichia coli.
Core Innovation
The invention relates to recombinant polypeptide constructs comprising enterotoxigenic Escherichia coli (ETEC) fimbrial subunits. These constructs contain multiple subunits from the same or different ETEC fimbrial types connected via polypeptide linkers and stabilized by donor strand complementation. The recombinant polypeptide constructs are designed to be included in immunogenic formulations to induce immunity against ETEC strains.
The problem addressed by the invention is the need for broadly protective immunogens against diverse ETEC strains that produce different fimbrial types. ETEC-mediated diarrheal disease is caused by bacterial adherence to intestinal cells via fimbriae, which are composed of major and minor subunits. Natural fimbriae contain minor subunits in low abundance, and these minor subunits are susceptible to proteolytic degradation outside the fimbrial structure. Thus, there is a technical challenge to develop stable, immunogenic constructs incorporating multiple fimbrial subunits that provide broad immunogenic coverage and are suitable for immunization.
The invention solves this problem by using recombinant polypeptide construct designs that connect major and minor fimbrial subunits from the same fimbrial type stabilized by donor strand complementation, which protects the subunits from misfolding and proteolytic degradation. Furthermore, multiple such constructs can be connected via linkers to form multipartite fusions containing subunits from different fimbrial types, including class 5a, 5b, 5c as well as CS3 and CS6 fimbriae. These multipartite fusions allow broader spectrum coverage and simplify manufacturing and administration.
Claims Coverage
The patent includes multiple claims primarily focusing on recombinant polypeptide constructs comprising ETEC fimbrial subunits and their various configurations. The key inventive features relate to the composition, stabilization, linkage, and multifunctional fusion of these subunits.
Recombinant polypeptide construct comprising fimbrial subunits connected via polypeptide linkers
The construct contains a whole or immunogenic fragment of E. coli fimbrial minor or major subunits connected to one or more major fimbrial subunits of the same fimbrial type via polypeptide linkers. Each major fimbrial subunit contains a donor β strand and is connected to the adjacent subunits via linkers. The C-terminal major subunit is connected to a donor β strand derived from a homologous or heterologous major subunit and can include a C-terminal histidine tag.
Incorporation of fimbrial types from various ETEC fimbriae
Fimbrial types include class 5a, 5b, 5c, CS3, and CS6 from ETEC. The construct can include subunits from any of these types to broaden immune coverage.
Donor β strand length and stabilization features
Donor β strands connecting the C-terminus have lengths of 12 to 16 amino acids. Constructs may contain 18-22 amino acid signal peptides at the N-terminus and deletions of 14 to 18 amino acids at the N-terminus of one or more major subunits to prevent undesirable associations and proteolytic cleavage.
Multipartite fusion constructs combining different fimbrial types
The recombinant polypeptide construct can be connected to one or more other such constructs containing fimbrial subunits from different fimbrial types through glycine residues as linkers. Each fimbrial type module is stabilized via donor strand complementation. The multipartite fusion can have C-terminal histidine tags.
Chimeric molecules with bacterial toxins
The construct can be connected, via a polypeptide linker, to bacterial toxins such as cholera toxin A2 subunit forming chimeric molecules. These chimeras are noncovalently associated with bacterial toxin multimeric compositions like LTB5 for holotoxin-like structures.
Specific fimbrial minor and major subunits incorporated
Minor subunits can be from CfaE, CsfD, CsuD, CooD, CsbD, CosD, CsdD, CotD; major subunits from CfaB, CsfA, CsuA1, CsuA2, CooA, CsdA, CosA, CsbA, CotA, CstG, CstH, CssA, CssB or their immunogenic fragments or derivatives.
Polypeptide linker compositions
Linkers used in the constructs can be SEQ ID No. 5, tri-glycine, or other suitable amino acid sequences enabling flexibility and reducing proteolytic degradation.
The independent claims cover recombinant polypeptide constructs comprising ETEC fimbrial minor and major subunits connected by polypeptide linkers and stabilized by donor β strands, optionally formed as multipartite fusions incorporating multiple fimbrial types and chimeric toxin fusions to induce broad immunity against ETEC strains.
Stated Advantages
The constructs provide broad immune coverage against multiple ETEC strains by incorporating multiple fimbrial subunits from different fimbrial types in a single molecule.
Donor strand complementation stabilizes the fimbrial subunits, preventing proteolytic degradation and misfolding, improving immunogenicity.
Multipartite fusion constructs simplify manufacturing and standardize administration over multiple single-component vaccines.
Inclusion of chimeric toxin components enhances immunogenic response through holotoxin-like structures.
Documented Applications
Use of recombinant polypeptide constructs as immunogenic compositions such as subunit vaccines to induce immunity against ETEC strains in mammals including humans.
Administration methods include oral, nasal, subcutaneous, intradermal, transdermal, sublingual, intramuscular routes.
Use of constructs expressed from recombinant DNA inserted into bacterial, viral or plasmid vectors for live vaccines.
Use of recombinant host cells transformed with DNA encoding the constructs to produce whole cells for vaccination against ETEC.
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